Project Summary/Abstract Natural products form a validated and preeminent source of new drug leads, but the low rate of new discoveries and the limited access of bioactive compounds are challenging current natural products-based drug discovery and development. The overall goal of the PI?s research program is to access and expand chemical diversity of microbes by the use of innovative strategies, thereby advancing natural products research. The central hypothesis of this work is that microbial genomes can be exploited to explore natural products and analogs. Bioinformatics mining of biosynthetic gene clusters from thousands of microbial genomes have distinguished microbes as the prominent source of an immense number of new therapeutically relevant compounds. However, current discovery process is able to access only a small fraction of these compounds, underlining the urgent need of new strategies. Enzymes as biocatalysts are proven alternatives for chemical synthesis in academic and industrial settings. The biosynthesis of microbial natural products is enriched with functionally diverse enzymes. Discovery of new enzymology from microbes can advance the synthesis of structural complex natural products and analogs via chemical methods or biological approaches (e.g., synthetic biology). To achieve the goal and test the hypothesis, research direction 1 of this proposal will focus on producing cyanobacterial natural products using multiple synthetic biology chassis. Cyanobacteria are a prolific source of structurally and functionally diverse natural products, but almost all cyanobacterial compounds are accessed only through the isolation from collected field samples. The research direction 1 can lead to the robust, thorough exploration of cyanobacterial strains directly from the genomes. Research direction 2 of this proposal will discover and characterize synthetically significant enzymes from microbial BCs, aiding the access and expansion of chemical diversity of natural products. The new enzymes discovered in direction 1 will also be characterized in direction 2 and can then be used in direction 1 to produce natural and unnatural compounds, demonstrating the improved cost efficiency. Specific attention in direction 2 will be to discover and characterize novel nitration enzymes. Together, these two directions of the PI?s research program can afford innovative strategies that enable effective exploitation of the seemingly limitless chemical diversity of microbes for discovery and development of new drugs and allow the development of new chemical processes. These studies can also boost the transformation of natural products research from small-scale pursuits to a genome-based high-throughput endeavor, therefore potentially triggering a paradigm shift in natural products and drug research.